The long-term goal of the proposed research is to define the mechanisms through which the TRP channels in photoreceptor cells are activated and regulated in response to light. The current proposal focuses on Drosophila phototransduction, which functions through a phospholipase C (PLC)-dependent signaling system, and culminates with Ca2+ and Na+ influx, via the TRP and TRPL channels. There exists a large family of mammalian TRPs, including channels in the intrinsically photosensitive retinal ganglion cells (ipRGCs) that are gated through a cascade that has notable parallels with fly phototransduction. The specific goals of the current proposal are to answer major questions in Drosophila phototransduction concerning the mode of activation and regulation of the TRP channels. To accomplish our goals, we will employ a multidisciplinary approach, using a combination of molecular genetics, biochemistry, cell biology, and electrophysiology. The goal of aim 1 is to identify the molecule that directly gates the TRP and TRPL channels. Prior to activation of these channels, PLC causes hydrolysis of PIP2 to generate IP3, DAG and H+. However, despite the >20 years that have elapsed since the identification of the Drosophila TRP channels, the precise activation mechanism is not known. We recently identified a DAG metabolite that increased in concentration in a light-dependent manner. In aim 1 we will test the hypothesis that this is the agonist that gates the light-sensitive channels. The goals of aims 2 and 3 are to characterize candidate regulatory subunits that influence the activity and/or localization of TRP. Many ion channels associate with regulatory subunits. However, there has been no systematic attempt to identify proteins that interact with and modulate Drosophila TRP in photoreceptor cells. We affinity purified the TRP complex from flies, performed mass spectrometry and identified several candidate TRP regulatory proteins. In aim 2, we propose to test the hypothesis that another large transmembrane protein modulates TRP. In aim 3 we will test the hypothesis that two previously uncharacterized single transmembrane proteins that associate with TRP are required for the localization and activity of the channel. Aim 4 will test the PCaM model, which would explain how inhibition of TRP by calmodulin is coupled to phototransduction. According to this model, phosphoinositides (PIs) bind to TRP and preclude its association with calmodulin. Following light stimulation, PI levels decline allowing calmodulin binding and negative feedback. We suggest that the proposed studies are significant because they offer to resolve the mechanisms by which the TRP channels in photoreceptor cells are gated, localized and regulated. We also suggest that these studies will provide the framework for answering similar questions relevant to the channels in the ipRGCs, which contribute to light- induced circadian rhythms, sleep patterns and rudimentary image formation in the absence of rods and cones.